The epidemic of gonorrhea (2 million cases/year in U.S.) has made Neisseria gonorrhoeae one of the most important human pathogens. The cell surface polymers of the gonococcus, which have been implicated as virulence factors, show considerable phenotypic variation in expression. In this proposal we use a combination of biochemical, genetic, and recombinant DNA techniques to increase our understanding of the mechanisms controlling surface variations in the gonococcus. We are in the process of isolating specific gonococcal genes by recombinant DNA techniques. We have constructed an E. coli/N. gonorrhoeae shuttle vector, pLES2, that enables us to isolate cloned genes in E. coli and return them to N. gnorrhoeae without subcloning. These cloned genes will be used to generate a map of the gonococcal chromosome by chromosome walking, to determine DNA sequences, to construct merodiploids and analyze genes by cis/trans and dominant/recessive tests, to supply templates for in vitro transcription/translation, and to measure in vivo mRNA levels by hybridization. In addition, we are attempting to increase our means for genetic manipulation in N. gonorrhoeae by introducing a system for transpositional mutagenesis, by introducing the mucAB genes to increase mutability of the gonococcus, and by constructing gonococcal Hfrs by cloning the tra region frm a gonococcal conjugal plasmid into a gonococcal suicide vector. Finally, we are examining the surface antigens produced by the gonococcus when it is grown anaerobically, to determine if the organism expresses new virulence factors under these conditions.